Description: (Abstract of the project) Tight junctions seal the paracellular space between renal epithelial cells allowing maintenance of electroosmotic gradients required for absorption and secretion. Transient renal ischemia results in breakdown of the tight junction paracellular barrier and loss of normal renal function. Occludin is the only known transmembrane protein of the tight junction and preliminary studies suggest it functions in creating the paracellular seal. The overall goals of this application are to determine the molecular basis by which occludin creates a paracellular seal, how the barrier properties are regulated by cytoplasmic components and signaling pathways and how occludin responds to reversible renal ischemia. We have recently shown occludin is an intercellular adhesion molecule and we will use full-length and mutated occludin expressed in both cultured fibroblasts and renal epithelial cell lines (MDCK and LLC-PK1) to determine the basis for adhesion. Peptides corresponding to portions of the two extracellular domains of occludin will be used to localize the regions most responsible for adhesion. Constructs lacking either the first or second loop will be expressed to determine which is most involved in adhesion. This information will be translated to cultured epithelial lines to determine if adhesion is the basis for creation of the paracellular seal as measured by transepithelial electrical resistance and the paracellular flux of radiolabeled tracer molecules. As a non-barrier function of occludin, we observe its expression in fibroblasts inhibits cell motility. A role in motility may be important in epithelial restitution following ischemic damage of the tubular epithelium. We will determine whether adhesion is the basis for this motility regulation. The role of cytoplasmic factors such as actin organization ATP levels and signaling pathways in conferring adhesion will be investigated in fibroblasts and cultured epithelial cell lines. Finally we will study the structural and biochemical alterations to occludin occurring during ATP depletion and recovery in cultured renal epithelial cell lines and in ischemia and recovery in an intact rat kidney model. Specifically we will determine changes in the phosphorylation and extractability of occludin and correlate these with changes in the cellular localization of occludin determined by immunofluorescence, immunogold TEM, and immunogold freeze fracture electron microscopy. Together these studies will advance our understanding of how the paracellular barrier is created and regulated in renal epithelia and how the barrier is disrupted and reforms following during transient ischemic injury.